Blind Man’s Bluff – Elaboration of Fragment Hits in the Absence of Structure for the Development of Antitrypsin Deficiency Inhibitors
Stephen J. Headey A D , Mary C. Pearce B , Martin J. Scanlon A C and Stephen P. Bottomley B DA Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Vic. 3052, Australia.
B Biochemistry and Molecular Biology, Monash University, Vic. 3800, Australia.
C Australian Research Council Centre of Excellence for Coherent X-ray Science, Monash University, Parkville, Vic. 3052, Australia.
D Corresponding authors. Email: stephen.headey@monash.edu; steve.bottomley@monash.edu
Australian Journal of Chemistry 66(12) 1525-1529 https://doi.org/10.1071/CH13290
Submitted: 13 June 2013 Accepted: 1 August 2013 Published: 16 September 2013
Abstract
The three pillars of rational drug design from a fragment library are an efficient screen, a robust assay, and atomic-resolution structures of the protein–ligand complexes. However, not all targets are amenable to structure determination by X-ray crystallography or NMR spectroscopy. In particular, targets involved in diseases of protein misfolding are inherently intractable. In the absence of structures, we are blind. However, the lack of structural information need not preclude the use of fragment-based approaches. The use of appropriate NMR techniques can enable us to detect the effects of protein binding on ligand resonances. In our efforts to identify compounds that affect the kinetics of α1-antitrypsin misfolding, we have used saturation transfer difference NMR spectroscopy to detect hits from mixtures of compounds in a fragment library. In the absence of structures, the initial challenge is three-fold: to (1) distinguish between binding sites; (2) evaluate the relative affinities of hits; and (3) advance them to the stage where activity can be detected in biological assays. We largely achieved these aims by the use of Carr–Purcell–Meiboom–Gill NMR competition experiments that detect differential relaxation of the ligand on protein binding.
References
[1] B. Gooptu, D. A. Lomas, Annu. Rev. Biochem. 2009, 78, 147.| Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1MXos1Ghtbw%3D&md5=2af8a51e831e67518b6ab8f49ca762a1CAS | 19245336PubMed |
[2] I. Blanco, E. Fernández, E. F. Bustillo, Clin. Genet. 2001, 60, 31.
| Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BD3MvovFWhtQ%3D%3D&md5=11e4a8dada2e9edfd690bb6a97b784e2CAS | 11531967PubMed |
[3] D. A. Lomas, D. L. Evans, J. T. Finch, R. W. Carrell, Nature 1992, 357, 605.
| Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK38Xks1Oqu7w%3D&md5=fe7fe70438a9bb67a53fde519ac1a088CAS | 1608473PubMed |
[4] D. A. Lomas, D. L. Evans, S. R. Stone, W. S. Chang, R. W. Carrell, Biochemistry 1993, 32, 500.
| Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK3sXjsV2ltw%3D%3D&md5=7488cf654d76811f7790436a7f7f0da0CAS | 8422359PubMed |
[5] A. S. Knaupp, V. Levina, A. L. Robertson, M. C. Pearce, S. P. Bottomley, J. Mol. Biol. 2010, 396, 375.
| Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3cXht12murw%3D&md5=c6133ab3384678b47069a4f46907d19cCAS | 19944704PubMed |
[6] A. S. Knaupp, S. P. Bottomley, J. Mol. Biol. 2011, 413, 888.
| Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3MXhtlKisbnP&md5=d0ab615c99c23405dbb13594583ee7edCAS | 21945526PubMed |
[7] I. Kass, A. S. Knaupp, S. P. Bottomley, A. M. Buckle, Biophys. J. 2012, 102, 2856.
| Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC38XptVKrurY%3D&md5=1eb9e378f021f079e927599b46582a59CAS | 22735536PubMed |
[8] H. Koloczek, A. Banbula, G. S. Salvesen, J. Potempa, Protein Sci. 1996, 5, 2226.
| Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK28XntVKqtLk%3D&md5=4b9385a6789d1a8704afab2fc690a7cbCAS | 8931141PubMed |
[9] E. L. James, S. P. Bottomley, Arch. Biochem. Biophys. 1998, 356, 296.
| Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK1cXlsFCkuro%3D&md5=155f5bc1ba1b48d0079c10f55770693fCAS | 9705220PubMed |
[10] T. R. Dafforn, R. Mahadeva, P. R. Elliott, P. Sivasothy, D. A. Lomas, J. Biol. Chem. 1999, 274, 9548.
| Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK1MXisVWltbc%3D&md5=c88625347d7a4e171b591266e7306dddCAS | 10092640PubMed |
[11] G. L. Devlin, M. K. M. Chow, G. J. Howlett, S. P. Bottomley, J. Mol. Biol. 2002, 324, 859.
| Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD38XpsF2kur8%3D&md5=e255800f14ae207c8701e5746d9f8334CAS | 12460583PubMed |
[12] D. J. Tew, S. P. Bottomley, Biochim. Biophys. Acta 2001, 1481, 11.
[13] M. C. Pearce, C. J. Morton, S. C. Feil, G. Hansen, J. J. Adams, M. W. Parker, S. P. Bottomley, Protein Sci. 2008, 17, 2127.
| Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1cXhsVKgt7vP&md5=25d6b9ef55cf153031079e9882423657CAS | 18780818PubMed |
[14] G. L. Devlin, H. Parfrey, D. J. Tew, D. A. Lomas, S. P. Bottomley, Am. J. Respir. Cell Mol. Biol. 2001, 24, 727.
| Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3MXltVWhtr8%3D&md5=3fe3e2f15432d4cba8f14f0902e0fdeaCAS | 11415938PubMed |
[15] M. K. Chow, G. L. Devlin, S. P. Bottomley, Biol. Chem. 2001, 382, 1593.
| Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD38XktlOmtg%3D%3D&md5=ac6bf0a2f09b0db0350fff79632167c4CAS | 11767949PubMed |
[16] B. J. Stockman, C. Dalvit, Prog. Nucl. Mag. Reson. Spectro. 2002, 41, 187.
| Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD38XovFCjsLs%3D&md5=cb59315e6f77f79c2dc494258873c392CAS |
[17] M. Mallya, R. L. Phillips, S. A. Saldanha, B. Gooptu, S. C. Brown, D. J. Termine, A. M. Shirvani, Y. Wu, R. N. Sifers, R. Abagyan, D. A. Lomas, J. Med. Chem. 2007, 50, 5357.
| Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2sXhtFWksbbK&md5=440e30e6d2e0e6529fa49ef938ec878bCAS | 17918823PubMed |
[18] V. Levina, W. W. Dai, A. S. Knaupp, D. Kaiserman, M. C. Pearce, L. D. Cabrita, P. I. Bird, S. P. Bottomley, Protein Expr. Purif. 2009, 68, 226.
| Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1MXht1WmsL3J&md5=7413458b907ebbb3cfab6065a5e909b5CAS | 19555763PubMed |
